Apparatus for detecting target molecules and related methods

ABSTRACT

An apparatus for analysis of a sample and in particular of a biological sample. The apparatus contains a microfluidic chip with dies, adapted to be selectively activated or deactivated by presence of target molecules in the biological sample. The apparatus further contains a light source to emit light for illumination of the microfluidic chip and an optical filter to allow passage of the light from the dies once activated or deactivated by the presence of the target molecules. A method for pressurizing a microfluidic chip is also disclosed, where a chamber is provided, the chamber is connected with the microfluidic chip and pressure is applied to the chamber.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. application Ser.No. 11/856,722 filed on Sep. 18, 2007, which claims priority to U.S.Provisional Application entitled “Medical Diagnostic Tool”, Ser. No.60/845,336 filed on Sep. 18, 2006, the disclosure of which isincorporated herein by reference in its entirety. This application maybe related to U.S. application Ser. No. 11/804,112, docket numberP002-US, entitled “Fluorescence Detector, Filter Device and RelatedMethods”, filed on May 17, 2007. This application may also be related toU.S. Application Serial No. 13/348,495, docket number P002-USC, entitled“Fluorescence Detector, Filter Device and Related Methods”, filed onJan. 11, 2012.

TECHNICAL FIELD

The present disclosure relates to detection of target molecules andmicrofluidics. In particular, it relates to an apparatus for analysis ofa sample and a method for pressurizing a microfluidic chip.

SUMMARY

According to a first aspect of the present disclosure, an apparatus foranalysis of a sample, and in particular a biological sample, isdisclosed, the apparatus comprising: a microfluidic chip containingdies; a light source to emit light for illumination of the microfluidicchip; an optical filter; and a detector to detect the light passingthrough the optical filter, wherein presence of target molecules in thebiological sample activates and/or immobilizes a die at a positiondetectable by the detector, and the optical filter allows passage of thelight from the dies once activated and/or immobilized by the presence ofthe target molecules.

According to a second aspect of the present disclosure, a method forpressurizing a microfluidic chip comprising sealed fluidic circuits isprovided, comprising: providing a first chamber in the microfluidicchip; and applying pressure to the first chamber.

According to a third aspect of the present disclosure, a method torelease liquids in a microfluidic circuit is provided, comprising:introducing a liquid in a first chamber inside the microfluidic circuit;sealing the chamber; connecting the first chamber to a first channelthough a sacrificial membrane; providing a second chamber; connectingthe second chamber to the first chamber through a second channel; andcompressing the second chamber to release the liquid from the firstchamber into the first channel.

Further embodiments of the present disclosure are shown, in thespecification, drawings and claims of the present application.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description, serve toexplain the principles and implementations of the disclosure.

In the drawings:

FIG. 1 is a schematic illustration of an apparatus for analysis of asample in accordance with an embodiment of the present disclosure.

FIG. 2 shows an example of a lock-in amplifier to be used together withthe apparatus of FIG. 1.

FIGS. 3A and 3B show a schematic illustration of a passive CMOS pixelarray (FIG. 3A) and active CMOS pixel array (FIG. 3B) to be used asimagers in the embodiment of FIG. 1.

FIG. 4 shows an exemplary mixing technique for use with the embodimentof FIGS. 3A and 3B, where a mixer is implemented by applying a referencesignal to a switch.

FIG. 5 shows a variation of FIG. 4, where a filter network and anintegrator are further provided.

FIG. 6 shows a further variation of FIG. 4, where a switched capacitorsampler-buffer circuit is used.

FIG. 7 shows an example of an architecture for the imaging circuit.

FIGS. 8A-8D show examples of sealing mechanisms for a sample chamber ofa microfluidic chip.

FIGS. 9A and 9B show further embodiments of the sealing mechanism forthe sample chamber.

FIGS. 10 A and 10B show a valve used to seal a reactant chamber or togate propagation of the liquid through the chamber. The gating occurs bypressure threshold. Every time the pressure exceeds a new threshold anadditional gate (valve or sacrificial membrane) opens and the liquid canpropagate further.

FIGS. 11A and 11B show examples of mechanical actuation of themicrofluidic chip.

FIGS. 12A and 12B show actuation of the microfluidic chip with pressurecontrol.

FIG. 13 shows a mechanism to push sample liquid through a filter andinto a channel in the microfluidic chip. According to a firstembodiment, a piston pushes on an air filled chamber connected to thesample or reactant chamber with a microfluidic channel. However, afurther embodiment is also possible, where the piston pushes directly onthe sample/reactant chamber. Similar considerations apply to FIGS. 14and 15.

FIG. 14 is a schematic illustration of a mechanism to push reactant intoa channel according to an embodiment similar to the one disclosed inFIG. 13.

FIG. 15 shows an embodiment of a rail system and sample chamber sealingmechanism.

FIGS. 16A and 16B show further embodiments of the circuit of FIG. 5.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

In accordance with a first embodiment thereof, the present disclosurerelates to an apparatus for detecting target molecules that can be usedas a diagnostic tool or apparatus to analyze biological samples, forexample to detect small quantities of proteins with an Enzyme-LinkedImmunoSorbent Assay (ELISA), as well as several methods that can beapplied to the apparatus, for example, in order to make it moreperformant, more user friendly, or more marketable. The apparatus is notrestricted to ELISA, but is generally applicable to detection mechanismsbased on a microfluidic chip or optical detection. Regardless of thespecific biochemistry used, the applicants introduce a series oftechniques that enable, amongst other things, user friendly actuation ofmicrofluidic devices, cheap manufacturing, high sensitivity for opticaldetection schemes, multi-purpose usage of the tool (that is withdifferent types of microfluidic chips and for different types ofanalysis) and, in medical related applications, efficient communicationwith doctors, insurances, pharmacies etc.

FIG. 1 shows a high-level schematic of the apparatus in accordance withthe disclosure. Light is emitted from a light source (1), e.g. a brightLED or a laser diode. The light illuminates portions of a microfluidicchip or apparatus (3) that contains dies (9). Optionally, between thelight source (1) and the microfluidic chip (3), an optical filter (2)can be placed that leaves the wavelength that excite the dies (9) gothrough but that filters out the wavelengths emitted by the dies. Duringthe course of the biochemical analysis, the dies (9) in the microfluidicchip are selectively activated by the presence of certain molecules inthe biological sample, so that emission from the dies is a quantifiablesign of the presence of these molecules. In some detection methods,presence of the target molecule modifies a sequence of events so as toactivate a die (e.g. ELISA or other enzyme linked assays). However inother types of assays (for example in typical DNA microarrays or otherfluorescently linked assays) the presence of the target moleculemodifies a sequence of events to immobilize the die at a certainposition. The mechanism of this activation is often an indirect one,where the presence of the detected molecule (the target molecule) willgive rise to a series of reactions that will activate the dies. That is,the dies (9) are modified from a molecule or state in which they don'temit light or emit light that is filtered out by filter (4) to amolecule or state in which they emit light that can be transmittedthrough (4). In the following it will also be understood that theconverse mechanism is possible. A chain of events triggered by thetarget molecule could also deactivate the dies. In that case thedeactivation and not the activation of the die will be detected as aquantifiable signature of the target molecule.

This occurs, for example, when proteins are detected with an ELISAstack. A second optical filter (4) is ideally a notch-filter (narrowpass) that only leaves through light corresponding to the emissionwavelength of the dies, or a filter that leaves through light emitted bythe dies, but none of the light that is not blocked by filter (2). Oneor several lenses (5) can image the microfluidic circuit on a detector(6) such as a CCD or a CMOS imager. In some embodiments, a photodiode, acharge coupled device, a phototransistor or a photomultiplier tube canalso be used. Optionally, reflectors (7) can be included in theapparatus to reflect back light that would otherwise not be absorbed bythe dies (for example dichroic minors), in order to excite them with asmuch light as possible. The light source (1) can be modulated, forexample by means of electric direct modulation, or by using a mechanicaldevice such as a chopper, or by using another type of external modulatorsuch as for example a Pockels Cell. A lock-in amplifier (not shown inFIG. 1) can then be used to detect signals with very bad signal to noiseratios. The lock-in amplifier can be on the same chip as the detector(6), for example if the detector is a CMOS imager and the lock-in madein CMOS technology. The same chip can also electrically modulate thelight source through connection (8).

The apparatus herein described can comprise other elements, some ofwhich are described in details herein below. In particular, in someembodiments the apparatus is very compact and, in embodiments wherein adiagnostic analysis is desired, can take the form of a hand-helddiagnostic tool.

In particular, in some embodiments, the apparatus provides the means forpatients to monitor the emergence of medical conditions such as cancersor other diseases, as well as to monitor the development of a knowncondition, or the results from a treatment, by monitoring specificmolecules such as proteins in biological samples such as blood orsaliva. The apparatus can be at the patients' home so that these testscan be made more frequently, more conveniently and at a smaller costthan if the patient had to go to a specialized laboratory every time.This is frequently described as personalized medicine.

The embodiments of the diagnostic device herein described can comprisemeans to access an Internet portal or a remote server. In thoseembodiments, data can be exchanged between the apparatus and theInternet/Remote Server, for example by means of either:

-   -   a dial-up or other internet modem (cable, DSL etc.) directly        incorporated into the tool.    -   an Ethernet port incorporated into the tool that permits        plugging the tool into a local area network (LAN).    -   a Wi-Fi or other wireless device incorporated in the apparatus        that enables connectivity with a LAN.    -   a Blue-Tooth connection or another wireless protocol        incorporated into the apparatus that enables the apparatus to        communicate with a computer or with a cell phone. The computer        or the phone then provide the connectivity to the Internet or to        the remote server.    -   a connection with a computer for example by means of a USB port        or other ports/protocols that enable data transfer such as        Ethernet, Firewire etc. The computer then provides the        connectivity to the Internet or to the remote server.    -   a connection that enables the exchange of data with a phone. The        phone then provides the connectivity to the Internet or to the        remote server.

In some embodiments, the portal can enable the patient's doctor toaccess remotely the results of the diagnostic tests. In some embodimentsthe portal can also permit the doctor and the patient to obtaininformation about drugs relating to specific medical conditions. In someembodiments, advertisements for relevant drugs can be posted through theportal. In some embodiments, the portal enables large-scaledata-collection over large population of patients. This data-collectioncan be used for example to evaluate medical trials or for statisticalpurposes by entities such as insurances or public-health organizations.The portal can guarantee the privacy of the patient during suchdata-collection by compiling the data and only delivering a compiledreport, devoid of private information, to the receiver of the report. Insome embodiments, the portal can also enable patients and doctors toorder drugs or buy other health related items online, such as bookscovering the topic etc. In some embodiments, the portal can be used tosend patients reminders about scheduling of health related items (suchas taking your medicine or going to see your doctor), it could warn thepatient that a potentially critical condition (detected by thediagnostic device) warrants a visit to a doctor. The agendafunctionality would be particularly powerful if the patient needs orwishes to use the diagnostic device on a regular basis, but also needsor wishes to take care of other health related tasks that are eitherinfrequent or very diverse. In some embodiments, the portal can enableother communication channels between the patient, the doctor, theinsurance, the pharmaceutical industry, drug distributors and otherproviders of health care related items and services.

The portal might be used also to exchange communications between usersin embodiments wherein the target detection is associated withnon-medically related uses of the device herein described. A skilledperson will be able to identify the non-medical related embodiments ofthe portal herein described, which will not be described herein indetail.

In some embodiments the apparatus includes a detector array There aremany advantages of using a detector array, such as a CCD array or a CMOSimager, rather than a single photodetector or a small number ofphotodetectors. For example, with a detector array the position on themicrofluidic chip from which the light originates can be determined.Therefore, several different types of tests can be done on the samemicrofluidic chip even if they all activate dies emitting at the samewavelength. The discriminating information is then provided by spatiallocation, that is depending where on the imager the fluorescing dies areimaged provides the apparatus with the information which protein hasbeen detected. For example, when an ELISA stack is used to detectproteins, different proteins can be detected with different antigen orantibodies, but the stack corresponding to the detection of differentmolecules can activate the same die.

In some embodiments wherein a large number of pixels is involved, eachindividual pixel can be connected to its own amplifier (e.g. a lock-inamplifier). In some embodiments wherein a large number of pixels isinvolved a circuit that would otherwise be duplicated for each pixel canbe divided into several pieces or stages, with a first circuit pieceduplicated for each pixel ad a second circuit piece common to all orlarge group of pixels. A further advantage associated with the circuitdivision is that it enables to minimize the duplication of electronics,with minimal impact on the chip size, routing and pixel responsivitywhile leaving crucial elements at each pixel. For example, it isbeneficial to integrate the signal of a pixel for a long time so as toaccumulate the signal but cancel out the noise (the noise grows as arandom walk and as such has a much slower accretion rate, so that thesignal to noise ratio increases over time). Moreover, it is beneficialto accumulate each individual signal independently so that high signalto noise data can be collected for all the pixels before it is known tothe chip which signals are relevant. Furthermore, propagating a signalthrough the optical detector chip can cause the signal to noise todegrade due to parasitic coupling to other parts of the chip and due tothe high capacitive load seen an amplifier located at the end of theline. However, in a lock-in configuration, where the signal is not DCbut centered around a carrier frequency, it is not possible toaccumulate the signal before mixing (demodulation) because it willaverage out to zero. After mixing, the signal is converted to a DCcomponent that can be accumulated. Furthermore, if a DC component ispropagated through the chip rather than an AC component, capacitivecoupling becomes much less of an issue. For these reasons, it isbeneficial to incorporate at least the mixer, and possibly anintegrating element such as a capacitor, into each pixel. This can bedone for example by adding a switched capacitor, mixed-signal integratorto each pixel. Moreover, such method is compatible with a CMOS process.

As anticipated above, in order to detect molecules with very lowconcentration in a sample and in particular in a biological sample,lock-in amplifiers can be used. Lock-in amplifiers can recover a signaleven in the presence of an overwhelming noise background, thus extremelylow signals that would otherwise be lost in the noise can be recovered.

A lock-in amplifier is shown in FIG. 2 and comprises at least a mixer(12), a low-pass filter (13) and an amplifier (10 or 14). An additionalbandpass filter (11) is usually present. The mixer (12) uses a referencesignal at the same frequency and the same phase as the input signal. Ifthe input signal and the reference signal are out of phase, a phaseshifter (15) shifts the reference signal and the input signal back inphase. This can happen, for example, if the electrical signal thatdrives the light source experiences a significant phase delay beforearriving at the light source, or if fluorescence lifetime imagingmicroscopy is implemented. Alternatively to a phase delay, a dual phaselock-in amplifier can be used.

FIG. 3A shows a passive CMOS array, where a simple transistor (16)typically acts as a switch to connect a pixel to a column read-out lineand then to a column buffer in the column decoder. FIG. 3B shows anactive CMOS array where the simple switch transistor is replaced by apreamplifier (15). These active CMOS arrays have been shown to lead tobetter signal to noise ratios and are the most commonly occurring CMOSimager. The concept of active CMOS arrays can be generalized to includeparts of the lock-in amplifiers. In particular, for the purpose of theapparatus of the present disclosure, the pixels do not need to be verysmall, so that some more circuitry can be added to each pixel withoutsignificantly reducing the ratio of the pixel size covered by thephotodiode (that is without significantly reducing the sensitivity).

A part of the lock-in amplifier can be distributed throughout the CCDarray or the CMOS imager. For example, the mixer (12) (see FIG. 2) canbe distributed throughout the CCD or the CMOS imager, so that eachindividual photodiode, or local group of photodiodes, is connected toits own mixer. Other parts of the lock-in amplifier such as filters andamplifiers can also be added to each pixel. It is preferential to use acircuit compatible with a cheap CMOS process. The mixer can beimplemented with either an analog multiplier, a digital switch or adigital multiplier.

In the following, the applicants will provide examples of a distributedlock-in amplifier based on switched capacitance circuits and switches(the switches can be controlled by either a digital or an analog signal;the input to the switch is analog in nature). Individual switches can beimplemented by a single transistor or by a more complex circuit.

FIG. 4 illustrates the general principle. A photodiode (6) is mixed byapplying the reference signal to a switch (17). Element (18) can be asimple transistor such as transistor (16) of FIG. 3A, or anamplifier/buffer like element (15) of FIG. 3B, that only applies asignal to the column readout if triggered by row-select. A capacitoraccumulates the photocurrent. FIG. 4 is intended to convey that eachpixel contains at least a switch or analog mixer, an integrating elementsuch as a capacitor (or as seen later more complex integrating circuts)and an element (18) that connects the integrator to the read-out line.

This circuit can take different forms as exemplified by FIG. 5 and FIG.6, where an accumulator (FIG. 5) and a switched capacitor sampler-buffer(FIG. 6) are used. In all three cases, further amplifier or filternetworks can be placed in the column decoder or later in the circuit.FIGS. 5 and 6 also show how these circuits can properly bias thephotodiode.

The circuit in FIG. 5 has the advantage of integrating the signal evenif row-select is off. The filter network (19) is ideally a high Qpass-band filter centered on the modulation frequency of light source.At least it filters out the DC component of the reference signal. Theswitch (20) acts as a mixer. Element (21) is an integrator (for exampleimplemented with a capacitor and a differential amplifier, as shown inthe figure). As such it acts both as the low-pass filter (13) of FIG. 2(it only accumulates the DC component over time) and as an amplifier(10) or (14) of FIG. 2. Element (22) can either be an addressableamplifier such as element (15) of FIG. 2, an addressable buffer, or asimpler switch such as a single transistor (16), as shown in FIG. 3A.Additional filters can be added to the circuit, for example after theintegrator (21). Additionally, a reset function can be added (to resetthe accumulator, for example by discharging the capacitor in thisimplementation). Offset cancellation could also be added in case thedifferential amplifiers show mismatch. This offset cancellation couldrely on a feedback loop. It could also calibrated at when the electronicchip is switched on, or calibrated only once after fabrication of thechip and before selling the chip.

The circuit in FIG. 6 relies on a sampler-buffer (24). In thisembodiment the sampler-buffer/amplifier comprises three switches, acapacitor and a differential amplifier (see, e.g., “Design of analogCMOS integrated circuits”, Behzad Razavi, Mc Graw Hill, 2000). Thefilter network (23) is ideally a high-Q bandpass filter, at least itfilters out the DC component of the signal. Element (25) can be anaddressable amplifier such as element (15) described above, or a simpleswitch such as element (16) described above. The filter network (26) isa low-pass filter. Filter network (26) can also be removed from thepixel and added later in the circuit. Offset cancellation can also beadded such as in the case of FIG. 5.

In the above circuits, the timing of the reference signals can bemodified a little (small delays) to achieve charge injectioncancellation (see, e.g., Razavi, cited above, page 427). Other switchedcapacitor circuits can be used in the active pixels. For example thesampler-buffer (24) in FIG. 6 can be replaced by a non-invertingamplifier such as shown in Razavi, cited above, page 432. In this way,gain is added to the pixel output. The integrator (21) in FIG. 5 can bereplaced by a switched capacitor integrator as described on page 440 inRazavi, cited above.

Part of the lock-in amplifier can be removed from the individual pixeland placed later in the signal path. In that case, it can be eitherrepeated for each column readout (which would still take much less spacethan if that part would be duplicated for each pixel as there are manypixels on a single column) or it could even be shared by severalcolumns. For example, if a group of columns (activated by a group of rowselects) are known to be imaging a specific reservoir of themicrofluidic chip corresponding to the detection of a specific molecule,the signals can be added (to a sum 1) before further processing. If adifferent group of columns is known to correspond to the imaging of asecond reservoir detecting a second molecule, the signals of this groupof columns can also be added (to a sum 2) and routed to a differentoutput of the adder circuit. Alternatively, the sum 1 and the sum 2 canbe time multiplexed on the same adder output. If a subset of columnscorresponds to several reservoirs, for example reservoirs that havedifferent y-positions but overlapping x-positions, the summation circuitcan add the set of columns corresponding to a given reservoir while atthe same time the row addressing circuit can activate the rowscorresponding to this reservoir. Then the summation circuit can do thesame operation for the next reservoir, cycle through some morereservoirs and then start over (while at the same time the rowaddressing circuits activates the proper rows in synchronization withthe summation circuit). The signals corresponding to the individualreservoir (chamber) can then be time-multiplexed on the same output, orbe multiplexed to different outputs. If the output of the multiplexingsummation device is still analog, later stages of the regular amplifieror of the lock-in amplifier can be implemented after the summationdevice. These later stages only need to be laid out once, or a verysmall number of times. Finally the signal is digitalized with an A/Dconverter. One example of a good sub-circuit to place at the end of eachread-out line, or between the summation device and the A/D converter, isa very high performance filter that could potentially take significantchip real-estate.

The addition of the signals of individual columns can be made dynamical(for example it can be reprogrammed depending on the type ofmicrofluidic chip used). If the signals from the pixels are notamplified with a lock-in, but rather with a simple amplifier, the sameprinciples can be applied. Part of the amplifier can be placed at eachpixel, part can be specific to a row, and part can be placed aftersignals from several rows have been dynamically added. This principlecan also be applied to any other circuit in the signal path, although itmakes most sense for analog and mixed-signal circuits. Afterdigitalization data treatment can be made by a sequential dataprocessing unit such as a DSP, so that massively parallel circuitry doesnot represent a problem anymore.

It could be useful in most cases to have at least one accumulatorassociated to each pixel, so that signals can be accumulated even whennot being read at the moment (that is even when read-out is notactivated by row-select). The integrator circuit (21) shown in FIG. 5 ofthe present disclosure or the switched capacitor integrator shown onpage 440 of Razavi are particularly suited for this when combined withthe filter network (19) that cancels out the DC component of the signal.The integrator then also effectively acts as a low pass-filter becauseonly the DC component created by the mixer (here a switch) will beaccumulated over time. The higher frequency component will yield a zerocontribution when accumulated over extended periods of time.

The filter network (19) of FIG. 5 can be left out if the mixer (12) ofFIG. 2 is a more ideal mixer than a switch (for example a digitalswitch). If a switch is used the mixer is non-ideal, in that the switchdoes not multiply the input signal with a sine-wave, but by a periodicfunction that also has higher harmonics as well as a DC component. Dueto this DC component in the multiplying function, DC components in theinput signal will also create a finite DC component in the output of themixer. As the DC component at the output of the mixer is amplified inlater stages, and the DC component in the input signal is not part ofthe “target signal”, it should be filtered out prior to the mixer.Because of the higher harmonics of the multiplying function, noise inthe input signal at these higher harmonics can also be down converted tothe mixer. Thus it can be beneficial if he filter network (19) also cutsoff higher frequency noise. Alternatively, a more complex switch can beused that acts as a buffer in the on state and as an inverter in the offstate, this then leads to a multiplying function without DC component.

FIG. 7 shows a schematic of the chip architecture. A circuit, such asfor example a lock-in amplifier, an integrator, or a regular amplifier,is split into three parts, (27), (28), and (29). Element (27) isdistributed in the CMOS array (here only 9 pixels are shown in order tosimplify the schematic), so that it has to be duplicated for each pixel.Advantages are that the signal can be integrated for every pixel at thesame time, that higher signal to noise ratios are achievable at higherspeeds. An entire column shares the second portion of the circuit.Finally, individual columns are summed together (summation circuitmarked by E) and the outputs sent to the third stage (29) of thecircuit. There are several outputs to accommodate, for example, asituation where several microfluidic chambers are monitored. Eachindividual output could correspond to a different chamber (for exampleto monitor 2 molecules, or to have a detection chamber and a referencechamber to compare to). Alternatively, several output corresponding toseveral reservoirs can be time-multiplexed onto the same output. E caneither be programmed once and stay constant over a sustained period ofoperation (static re-routing), for example in the case where the twochambers (i.e. the detection chamber and the reference chamber) areoffset diagonally (that is, the chambers do not share a common row or acommon column), or dynamically re-routed to accommodate reservoirs thatshare a subset of columns. Indeed, if, for example, the two chambershave common columns (but different lines), they cannot be read at thesame time. They need to be read sequentially, by changing the state ofthe row decoder and of the summation circuit. The output of element (29)is sent to an A/D converter, and the data then sent to amicro-controller via a digital bus (marked by a thick line, the arrowindicates the direction of data-flow). A reference signal generatorsends a reference signal to the light source and to the components ofthe lock-in in order to modulate the light source at the same frequencythan the reference signal of the mixers. The reference signal is sent tothe individual pixels and to the other parts of the circuitry(distributed lock-in amplifier) that necessitates it. A phase shifter(15) ensures that the signal detected by the photodiodes in the pixelarray is in phase with the reference signal fed to the mixers.

Note that the reference signal is distinct from the clock of the digitalelectronics (not shown in the figure), although it could be generatedfrom the clock. The reference signal could also be generated by avoltage controlled oscillator (VCO), an external quartz crystal, or byother means. The micro-controller controls the row decoder, thesummation circuit, and optionally the reference signal generator and thephase shifter (15). Finally, the micro-controller is connected via adigital bus to an I/O circuit that handles connection to other chips(and could be part of the micro-controller), or directly implements acommunication protocol to communicate with a computer or with theinternet, either by cable or with a wireless connection. Alternatively,the data can be stored in a memory (on chip, or on a separate chip inthe apparatus), and then be downloaded at a later point of time.

It is understood that the incorporation of subsets of the lock-inamplifiers into the pixel and then addition of the rest of the lock-inamplifier at a later point of the circuit can be implementedindependently of the specific architecture of FIG. 7, and that thearchitecture of FIG. 7 can be applied to pixel arrays withoutdistributed circuits. Even in the extreme case, where the imager where apassive CMOS imager or another type of imager the architecture can beused to obtain data corresponding to one or several microfluidicchambers, while being able to distinguish between these datasets.

FIG. 7 shows an architecture with a classical row decoder/column decoderstructure. However all the concepts can be easily generalized to otherpixel addressing schemes.

If the output of the individual pixel is a voltage, several rows shouldbe sequentially read even if they correspond to the same chamber. Thesummation device sums the signals from all the relevant columns at agiven point in time. It can also sum the signals from the relevant rowsby integrating the signals, or the digital signal processor can do thatafter A/D conversion.

Because it is usually easier to add currents then voltages, elements(27) and (28) can be chosen to encode the signal as current amplitudemodulation. For example, in FIG. 4, element (18) could be a simpleswitch or a current-current amplifier.

If the output of the pixel is a current, it is possible to read out allthe rows pertaining to a given chamber at the same time, as the currentssimply add up on the column read-out line. The implementation of thesummation device is also easier. If the element (18/22/25) is anamplifier, it can be chosen to be a current-to-current amplifier, or avoltage-to-current amplifier (if the previous stage outputs a voltage).The integrator, or the switched capacitor integrator can be implementedto output a current. Examples of pixels outputting a current onto theread-out line is given, for example, by FIG. 4 if element (18) isimplemented by a transistor in the same configuration as element (16) inFIG. 3( a). In order to achieve some current gain (18) can be replacedby a current mirror with the second branch connected to the readout-line.

Using a configuration as in FIG. 16A incorporating a mixer, a switchedcapacitor integrator (or regular integrator) and a circuit loading theread-out line with a current has the a advantage over using a plaincapacitor, because the pixel can be read without discharging theaccumulator. As such, the current status is obtained at every readout,but integration, and as such enhancement of signal to noise, is ongoing.Resetting the accumulator is triggered by an independent reset signal(not shown in figure). On the other hand, in a circuit where thecapacitor is directly connected through a switch to the read-out line,reading the circuit also empties the capacitor that accumulates thesignal.

FIG. 16B shows how the filter network of FIG. 16A can be replaced by asubtractor that cancels the DC component of the photocurrent. Thesubtractor/integrator in FIG. 16B can also be implemented with aswitched capacitor circuit in order to properly bias the individualcomponents of the circuit (such as for example the amplifier and thephotodiode). The integrator/subtractor can also replace the combinationof DC cancellation filter network and integrator in the other circuits.In the circuits described above that do not have an integrator, but thatdo necessitate a DC cancellation network, a regular subtractor canreplace the functionality of the DC cancellation network (for example ofthe capacitance necessitated in the DC cancellation network is toolarge). If the switch (68) in FIG. 16B is not perfect (for example ifsome of the current leaks when the switch is in one of its states), theimperfection can be compensated by adjusting the value of the twocapacitors in the integrator/subtractor circuit, to that effect one orboth of these capacitors could be implemented by a varactor or othertype of adjustable capacitor. Alternatively to adjusting the capacitors,the duty ratio (that is the ratio of on-time to off-time) of thereference signal controlling the switch (68) can be adjusted.

In FIGS. 16A and 16B, (67) is a circuit used to properly bias thephotodiode, (68) is a switch, (69) an integrator/subtractor and (70) acircuit that converts the voltage output of (69) to a current that isrouted on the read-out line when row-select is on. (70) is optional asthe voltage output from 69 could also be directly routed on the read-outline via a switch/buffer, however a circuit like (70) enables thesimultaneous read-out of multiple rows and adding the signals. Apossible implementation of (71) is shown with two switches and aninverter (the inverted signal could also be provided by the row decoderto simplify the pixel electronics). In this embodiment the bias voltage(71) is chosen so that the transistor shown on the right side is offwhen its gate is connected to (71). More complicated circuits with thesame functionality as (70), that ensure lower noise, higher linearity oradditional gain, could also be used. In FIGS. 4, 5, 6, 16A and 16B linesthat are unlabeled and left unconnected on one end correspond to a biasvoltage.

There are definite advantages in terms of signal integrity and signal tonoise in doing a summation of the signals of the individual pixelbelonging to the same chamber before A/D conversion. For example, suchembodiment is advantageous if the signal of an individual pixel is belowthe noise floor of the A/D converter. If all the pixels are summedbeforehand the noise of the A/D converter will be applied only one tothe overall signal (instead of sqrt(n) times, where n is the number ofpixel; the square root is due to the fact that noise adds upincoherently). The summed signal can also be way above the noise floorof the A/D converter even if an individual pixel is not above the noisefloor. This also applies to the rest of the circuitry. For example, thenoise arising from elements (28) and (29) of FIG. 7 will only be appliedonce to the signal resulting from a group of pixel, while the noise ofelement (27) of FIG. 7 will be applied to all the pixels individually.For this reason, the designer should export noisy parts of the system toelement (28) and to element (29), if possible. For this reason, it mightbe beneficial to reduce element (27) to the bare minimum, with a mixer,an integrator (for example a capacitor) and a transistor controlled bythe row decoder to connect the output of the integrator to the read-outline.

The summation circuit 1, as well as the row addressing circuit and thecolumn decoder, might have to perform quite complex tasks as describedabove. However, each microfluidic chamber should be monitored oversustained time periods in order to obtain quantitative data (and theconcentration of a molecule rather than just its presence) so that thesequence of tasks should be repeated. Instead of sending commandsthrough the bus for every individual task, the sequence of tasks can beprogrammed into these circuits by the micro-controller before thebeginning of the monitoring. The circuits can then cycle independentlythrough the tasks. Because—in accordance with this embodiment—thecontrol part of the circuitry is digital, the individual elements willautomatically stay synchronous if started at the same time.

Finally, elements (28) and (29) of FIG. 7 can be combined (at thelocation of element (28) in FIG. 7), the A/D placed before the summationcircuit, and the analog/mixed signal summation circuit replaced by adigital summation. In such a case, the mixer could also be replaced by adigital multiplier placed directly after the A/D or after digital signalsummation. Of course, the person skilled in the art will understand thatall the digital functionalities can be handled by either a dedicatedcircuit or by the micro-controller.

All these concepts are applicable to different types of imagers. Forexample, the CMOS photodiode array can be replaced by an array ofavalanche photodiodes to achieve higher sensitivity. BiCMOS is atechnology that is particularly promising for this application. Theability to incorporate bipolar transistors enables much lower noiseamplifiers in a BiCMOS process as compared to a purely CMOS process.Moreover, growing a silicon thin film on top of the silicon wafer ispart of the BiCMOS process. This enables complex vertical implantprofiles (this is the reason why the silicon epitaxy is incorporated inBiCMOS in the first place, as the bipolar transistors necessitate thecomplex vertical implant profile). The bulk wafer can be chosen to havea suitable doping. A series of implants can then be made into this waferbefore epitaxy. Then the silicon film is grown with another initialdoping. Finally, a series of implantation steps can be made afterepitaxy. The availability of complex vertical implantation profiles isextremely useful for the design of high-performance avalanchephotodiodes. For example, a highly doped film at the very bottom of thediode is good to reduce series resistance (that gives rise to thermalnoise and increases the necessary bias to achieve avalanche underillumination). These complex doping profiles can be obtained in a BiCMOSprocess because implants can be made before epitaxy at a lower levelbelow the final silicon surface.

The embodiments described above, related to the chip architecture andcircuit architecture, can also be applied to a SiGe process. In a SiGeprocess, lower noise amplifiers than in silicon technology can bedesigned. Also, the absorption cross-section of light can be enhancedleading to even higher sensitivity. Finally, the avalanche effectrequires smaller voltages in SiGe than in Silicon. A particular exampleof SiGe process that could be used is a CMOS SiGe process or a BiCMOSSiGe process.

For CCD and CMOS imagers used in color cameras, thin film depositionmethods have been developed to incorporate filters on top of theindividual photodiodes or CCD devices. These filters are typically notchfilters in the optical domain that let pass either red, green or blue,so that color information can be recovered by correlating the detectedintensities of three adjacent pixels, one for each filter type. The sametechniques can be applied to deposit the filter (4) of FIG. 1 on top ofthe chip (the order of the lens (5) and the filter (4) of FIG. 1 in thelight path can be inverted with no consequences on device performance).The filter (4) can be applied to either the microfluidic chip (3) or theimager (6) or be a separate element in between. Moreover, in oneembodiment, filter (4) of FIG. 1 can correspond to a multiplicity offilters placed in front of a multiplicity of pixels, the filter beingdifferent for different pixel. This opens the opportunity of usingdetection schemes with different types of dies by placing filtersadapted to a particular die in front of a pixel known to be imagingmicrofluidic chambers in which that particular die will be used. Inanother embodiment, most pixels have the same filter. However, a fewpixels in the mist of the first group of pixels can have no filter or adifferent filter and are sparsely distributed throughout the firstgroup.

This second group of pixels is used to do a calibration measurement usedto calibrate the measurement done by the first group of pixel. Forexample, the second group of pixels can be used to determine thestrength of the light source. In this case, the pixels of the secondgroup can be either unfiltered, or alternatively, filtered with a filterthat only lets the light pass that efficiently pumps the dies. Then thelight intensity detected by the second group corresponds exactly to thelight intensity exciting the dies. Finally, in a third technique, pixelscan be left unfiltered so that parts of the microfluidic chip can beimaged by the imager in order to monitor the progression of liquidsinside the chip.

In some embodiments methods and devices in the microfluidic chip orcircuit (e.g., the microfluidic circuit (3) of FIG. 1) are designed toaddress the following problems:

1. Actuating the microfluidic chip in a way such that the apparatusaround the microfluidic chip requires only a moderate complexity. Inparticular, actuation mechanisms are disclosed in the present writingthat pressurize liquids inside the chip without necessitating externalpumps.

2. Loading a sample (and in particular a biological sample) into themicrofluidic chip in a user-friendly way, and so that no external pumpsare required to push the sample through the switch.

3. Storing reactants on the chip so that truly small amount of reactantscan be used (by removing the need for an external pump, the volume ofreactants needed also becomes much smaller) and so that the release ofthese reactants into microfluidic channels can be actuated in veryrobust ways. The term “sample” refers to the incoming patient sample tobe tested, while the “reagents” can be stored as liquid or can be storedin any number of lypholized or gelified states and then reconstituted.

4. Enabling proper calibration of the data collected from themicrofluidic chip. In particular, methods and devices enabling propercalibration of the data, also enable gathering quantitative informationabout molecule concentration rather than merely detecting the presenceof a molecule.

5. Designing other devices and methods pertaining to the microfluidicchip.

Microfluidics is often described as a means to produce cheap analyticalequipment for the life sciences. The microfluidic devices or chipsthemselves are very cheap to produce and the amount of chemicalreactants needed to perform an analysis is very little. However, oneproblem is getting the reactants into the microfluidic channel. Whilethe amount of reactant inside the microfluidic chip is very little, muchmore reactant is needed if the reactant is pumped on chip through asmall pipe. In order to provide off-the-shelf microfluidic modules thatcan be plugged into the analytical device and in order to be able tohandle only extremely small volumes, thus avoiding waste of expensivereactants, other filling and storing methods are described hereinafter.Those modules can be plugged in and out to change the test or do a newtest.

In order to avoid adding complexity to the analytical device, such asthe necessity for several pumps, and in order to avoidcross-contamination, it is beneficial to provide to the user amicrofluidic chip with all the reactants already on chip in varioussealed chambers. An additional chamber is dedicated to the sample to beanalyzed (the sample chamber as opposed to the reactant chambers). Thesample chamber can be opened by the user, for example by removing ascrew top. The sample can then be introduced into the chamber and thechamber subsequently sealed again by the user, for example by screwingthe screw top back on. Sealing devices other than a screw top can beused, such as for example a plug. For the purpose of sealing the samplechamber, a sealing device can be imbedded into the microfluidic chip(the latter typically fabricated with PDMS with its inner channelscoated with other materials such as, for example, epoxite to enableELISA analysis or non-DNA sticking coating). This embodiment isillustrated in FIGS. 8A-8D.

In FIGS. 8A-8D, element (30) shows the material out of which themicrofluidic chip is made, for example PDMS. Element (31) represents asecond optional material that is coated inside the chamber. Element (33)is an embedded material for the purpose of creating a user-actuated,reversible sealing mechanism such as a screw top (32), a “clip-in”mechanism (34) (FIG. 8B), or a plug (35) (FIGS. 8C and 8D). In someembodiments a clamp can also be used as a sealing mechanism.

Alternatively, another sealing mechanism can be used as shown in FIGS.9A and 9B. In these figures, a flap (36), that can be made out of thesame material of the bulk of the microfluidic chip (37), or out of adifferent material, is attached to the chip on the left of FIG. 9A atthe time when the chip is purchased by the patient. A coating (38) canbe applied to facilitate later adhesion of the flap (36) with the chip(37). In an exemplary embodiment, the user (e.g. a patient using thedevice for diagnostic purposes) can introduce the sample (40) into thesample chamber (39), and then seals the chamber. The chamber is sealedby pushing down the flap, and then sealing occurs, for example, byapplying pressure, heat or UV light (depending on the materials and theadhesion promoter used). Alternatively, a thin “tape-like” layer can beinitially attached to the lower side of the flap (36) or on top of thesample chamber (on top of (38) or (39)). After introducing the sample,the user takes off this tape-like film, exposing an adhesion layer thatwas protected until then. Closing the flap and applying for examplepressure, heat or UV radiation will then seal the sample chamber. Thistechnique has the advantage that it is compatible with very small samplechambers, as the flap can be much wider than the chamber. In thisconfiguration, the size of the sample size does not make it harder toseal the chamber correctly.

The reactants are placed inside reactants chambers during manufacturingand theses chambers are sealed after filling. After manufacturing, themicrofluidic circuits can be frozen, if necessary, in order to preservethe reactants. A reactant chamber can either be directly connected witha microfluidic channel, in which case the liquid stays inside thechamber because it is frozen, until utilization when it is thawed andpressure applied to the reactant chamber to push the liquid into thechannel. Alternatively, the reactant chamber can be connected to amicrofluidic channel, but separated from this channel by a thin membrane(made, for example, of PDMS or another material embedded in the PDMS) ora sealed valve (the valve could, for example, be sealed just by naturalPDMS by way of PDMS adhesion, when closed). When a sufficient amount ofpressure is applied to the reactant chamber, the valve will open or themembrane rupture, letting the reactant flow into the channel. Thistechnique can also be applied to the sample chamber. Operation of amicrofluidic chip is described, for example, in U.S. Pub. App.2006-0263818, which is incorporated herein by reference in its entirety.

Some reactants are better pre-deposited on the surface of themicrofluidic channels or on the surface of the chambers in whichreactions will occur (the reactors). For example, an ELISA stackrequires the surface of the reactor to be coated with an antibody orwith an antigen. This coating should be made during manufacturing.

Sacrificial membranes or valves can be further used to control the flowof liquid in the microfluidic chip. The series of elements, valves ormembranes (FIGS. 10A and 10B), can be designed in such a way that thevalves or membranes open/rupture only when the liquid on one side isabove a certain pressure. This pressure can be made to be increasing forelements down the circuit, so that they can be opened in a sequentialorder and in a controllable fashion. Here the valves are meant toprovide a sealing mechanism. A property of the valve that is usuallyconsidered undesirable is used that is that the flap of the valve tendsto naturally seal with the other PDMS it gets in contact with,effectively sealing the channel.

FIGS. 10A and 10B illustrate the principle of a valve acting as a gatingmechanism. When the flap (45) is in the position shown in FIG. 10A, thevalve is closed. PDMS tends to stick to itself, so that when the valveis closed as shown in FIG. 10A, a pressure threshold needs to beovercome to open the valve. The pressure threshold can be set bymodifying the aspect ratio of the flap (45) and by modifying the area ofthe overlap of the flap with the lower part of the channel (the step inthe channel where the sticking occurs).

In some embodiments, particularly suitable for laboratory experiments,microfluidic chips are usually pressure-actuated by connecting theon-chip channels with a small pipe to an off-chip pump. In someembodiments, particularly suitable in applications, e.g diagnosticanalysis, to be performed out of lab (e.g. in the home an untrainedpatient) a completely sealed microfluidic chip (with the exception ofthe closure mechanism for the sample chamber that would have to beclosed by the user himself after filling) can be provided with simpleactuation mechanism that does not introduce undue complexity into theapparatus. This configuration would allow to minimize the complexityinvolved in connecting the device and the risks of cross-contaminationbetween samples usually associated with the presence of an open channeldirectly to the pump and to the associated possibility that reactantsand samples can flow to the pump and back to another microfluidic chipat a later test.

Accordingly, in some embodiments instead of applying pressure to theliquids in the microfluidic chip with a pump, in both sample chambersand reactant chambers the pressure can be applied by mechanicallypushing down on the chip with a piston. This is possible because thefluidic circuits are sealed (rather than left open to connect to thepump). However, it can be extremely difficult to pressurize very smallchannels in this way, because the mechanical actuator (such as a piston)is a blunt tool that pushes down an entire section of the chip insteadof precisely actuating a small chamber or a small channel. This could beremedied by connecting the small channel or the small chamber to a largechamber, the latter being pressurized by the piston. But again, this canbe an unsatisfactory solution due to the fact that now a large quantityof liquid is necessary in order to fill the large chamber—this liquidbeing possibly an expensive reactant or a sparse sample. This can beremedied by having a sealed microfluidic circuit (41) (see FIG. 11A)prefilled during manufacturing with a cheap liquid actuating themicrofluidic circuit (42) that is on the “signal-path”, that is,containing sample material or reactants etc. The circuit (41) can nowcomprise a large chamber (43) that is easily actuated by a mechanicalactuator (44), such as a piston pushed down by the apparatus onto themicrofluidic chip. The circuit (41) can be engineered to preciselyactuate the circuit (42), for example by pressurizing a chamberbelonging to (42) or by switching on or off a valve controlling the flowthrough a micro-channel belonging to (42).

These actuations schemes are illustrated in FIGS. 11A and 11B. In FIG.11A, the mechanical actuator pressurizes the control circuit (41) thatin terns pressurizes the controlled channel (42). The controlled channel(42) comprises a reactant chamber and possibly a sealed valve or asacrificial membrane between the reactant chamber and the rest of thecircuit marked as “To the rest of microfluidic circuit”. In FIG. 11B,the mechanical actuator (44) does not control the release of the liquidstored in a reactant chamber, but rather fluid flow later in themicrofluidic circuit. The actuator (44) operates by transferringpressure from the controlling circuit (41) to the controlled circuits(42 a) and (42 b). By changing the aspect ratio of the material betweenthe controlled and the controlling circuit, the pressure of the cuts ofeither of the controlled circuits can be set, so that the pressure afterwhich channels get cut off can be individually set even if they arecontrolled by the same channel.

FIGS. 12A and 12B illustrate advanced actuation schemes. Element (47)shows a mechanical actuator that pushes the piston onto the microfluidicchannel. Elements (48 a), (48 b) and (48 c) are sacrificial membranes orsealed valves that gate the progression of the liquid in the channel(42). (49 a), Elements (49 b) and (49 c) are sections that are not shownhere in which processing of the liquid occurs, for example, mixing withother reactants, heating, reactions etc. There are several ways in whichthe progression of the liquid through the individual gates can beorchestrated. Element (47) can be programmed to increment the pressureexerted on element (43) by predetermined amounts (or, alternatively, itcan be programmed to push the piston down by predetermined amounts).These predetermined amounts can be made dependent on the microfluidicchip inserted inside the diagnostic device (this is explained in detailin a following section). In order to do this, there should be either aposition or pressure sensor incorporated into actuator (47), or actuator(47) should be well characterized so that the micro-controller knowswhat sort of signal to apply to actuator (47) to obtain a given result.In these schemes, the actuator (47) should be continuously tunable ortunable in very small steps.

The cost of the actuator can be reduced if the actuator is set up sothat it is associated to a small and/or predetermined set of positionsthat can be the same regardless of what microfluidic chip is used andregardless of what test is performed. This result can be achieved byadapting the microfluidic chips rather than adapting the positions ofthe actuator. In some embodiments, this result can be in particularachieved, for example, by adapting the pressure at which the gatingelements let the flow pass, so that the pressure to open the gatecorresponds to the pressure that is created by the piston when it ismoved to its predetermined position. In some embodiments, another methodthat permits to use the same well characterized gating elements for allthe microfluidic circuits, is used. In particular instead of adaptingthe gating elements, the circuit between the gating elements can beadapted. For example, the channel can be made locally wider or thinnerbetween to consecutive gating elements, or a small chamber can be putbetween the gating element to modify the volume seen by the liquid (andthus modifying the pressure it will apply on the gating element given apredetermined piston position). If the piston is not set to go topredetermined positions, but to predetermined pressures, a small set ofgating elements (one for each pressure) can be directly applied to allthe microfluidic circuits without further adapting them.

If a feedback mechanism is incorporated in actuator (47), such actuatorcan also be used for monitoring purposes. For example, if the samplechamber is not properly sealed and the actuator is used to push a pistonon that chamber, or on a chamber connected to a microfluidic circuitused to control the sample chamber, the pressure change that will beobserved for a given position increment of the piston (and thus for agiven signal sent to actuator (47)) will be different from what isexpected. Compliance limits can be set for these pressure variations inorder to detect such events. On detection the device can warn the user,discard the measurement, or tag the measurement as being faulty.

If different pressures are necessary or desirable, that can also beobtained from a single actuator setting (that is the actuator is eitheron or off, pushing or not pushing) if several actuators are used thatare connected to separate control channel reservoirs (43 a) and (43 b)with different geometry (different top membrane size, different topmembrane thickness etc.). In this way, very simple actuators can be usedwith a small number of predetermined settings (for example just on andoff in the extreme case), and a multiplicity of pressures can still beobtained on chip, that can then be used to control various devices inthe microfluidic chip.

In some embodiments, an alternate way can be used to empty the contentof a storage chamber into a channel. This method is particularlyattractive for the sample chamber, as a substantial amount of air willusually be sealed inside the chamber along with the sample. Also, someclosure mechanisms such as the screw-top are quite bulky, leading to alarge chamber with rigid parts (the closure mechanism embedded in thePDMS) making it difficult to empty the chamber with the mechanismsdescribed above.

This is illustrated in FIG. 13. Sample liquid should be pressurized togo into thin channels such as channel (54). Additionally, otherobstacles might be on the sample path, such as for example a filter (52)to remove red blood cells and other non plasma component from the blood,as disclosed by Maltezos et. al. in U.S. patent application Ser. No.11/804,112 which is herein incorporated by reference in its entirety. Amechanical actuator (54) can push on the chamber, or on another chamber(55) filled with air and connected to the sample chamber to increase thepressure and press the liquid through the filter and into the channel.Because air is compressible, as opposed to liquid that is much lesscompressible, element (55) might need to be large relative to the amountof liquid it is pressurizing.

The same technique can be used for reactants as shown in FIG. 14. Inparticular, FIG. 14 shows a similar scheme applied to a reactant. Afterintroducing the reactant into the chamber, the chamber is sealed (56)during manufacturing. A sacrificial membrane (58) prevents any of thereactant to go into the channel prior to utilization of the chip by theend user. Typically, the reactant (57) will be frozen. However, toprevent the reactant to flow into the chamber (55) if it isinadvertently unfrozen while the chip is tilted or upside down, anadditional sacrificial membrane (59) is added. Both membranes (59) and(58) will only rupture when the actuator (54) compresses the chamber(55). Sacrificial membranes can be replaced by sealed valves.

Heaters can be incorporated into the apparatus to thaw the reactants,either all together, or in a predetermined sequence. In the latter case,heaters (in the forms of resistors) can be placed directly inside themicrofluidic chip (that is imbedded inside the PDMS), or placed outsidethe chip on the microfluidic “cartridge” (described below). Thawing thereactants in a predetermined sequence is a further means of controllingthe reactions leading to the detection of the targeted molecule.

The fabrication processes used to build the microfluidic channels can beused at the same time to fabricate cheap optics on the same chip. Forexample, chambers can be filled with (possible pressurized) liquidduring manufacturing, and then sealed, and designed to take the shape ofa lens, either naturally or under the action of the pressurized liquid.The index contrast between the liquid and the surrounding material(typically PDMS) then gives rise to the lensing effect.

Chambers can be filled and sealed with liquids during manufacturing thatare neither participating to the detection reaction itself, nor used toactivate other channels, but have a separate function all together:

A chamber can be filled with a die (activated—that is it does not needto undergo enzyme treatment to start luminescence as in the ELISA stack)that either emits light at the same wavelength then the die used in theELISA stack, or emits at a wavelength that the filter (4) also letsthrough. This die will then be imaged by the detector array (for exampleCCD or CMOS imager). This can be used for calibration purposes, forexample to normalize out device to device variations in the opticalpower emitted by the light source. Alternatively, a second detector canbe placed before filter (4), and its detected power used fornormalization. In this way, however, a second detector is not needed.

Furthermore, a chamber filled with such a die can be used as a lightsource that can be monitored by the detector (6) shown in FIG. 1. Thiscan be useful to monitor the progression of the testing process. Forexample, a microfluidic channel can be designed to be below such achamber, between the chamber and the detector. When fluid fills thecavity the amount of light received by the detector will change, and theimage seen by the detector will change (as the channel is illuminated bythe chamber). Thus filling of the channel can be detected.

A chamber filled with die that is imaged by the imager or detector (6)can also be used as a marker for position control (in order to tell theelectronics where exactly the microfluidic chip is located) or foridentification purposes (A given set of markers might identify a chip asa specific chip, the sequence of actuation and analysis the electronicsthen need to perform being programmed into the diagnostic device).

Also, these pre-activated dies can be used to calibrate the electronics,such as for example the offset cancellation in the lock-in stages, theamplifier gain etc.

Other problems can impact the reliability of the diagnostic tool, forexample variations in the ambient temperature that accelerate ordecelerate the reaction, or aging of the reactants. It is very useful tobe able to monitor these variables. For some applications, it is notonly important to be able to monitor whether a molecule is present, butalso its concentration in the sample and in particular a biologicalsample. In order to monitor this concentration, it is important toeither have tight control over the speed of the reaction activating theenzyme, or to be able to normalize out the other factors. Indeed in atechnique such as the ELISA stack, it is the time dependency of thefluorescence that reveals a concentration.

The calibration chambers can for example be the locus of an ELISAreaction with a liquid containing the detected antigen/antibody. Thefluorescence of that chamber, and the fluorescence ramp rate of thatchamber, can then be used as a calibration reference.

Alternatively, a calibration chamber can be the locus of a reactionbased on reactants with well characterized aging behavior that yieldsvariations in the fluorescence depending on the aging of the reactants.This can then be used to check whether the preemption date of the chipis past.

The calibration chamber can be the locus of a reaction that yieldsfluorescence detectable by the imager, the intensity and rate of changeof the fluorescence being very susceptible to temperature. This can thenbe used to normalize out the effect temperature will have had on theactual reaction that tests for the target molecule.

In some embodiments, a given microfluidic chip can only be used toperform a finite number of tests. After these tests, it will need to bediscarded and replaced by another microfluidic chip. To facilitateswapping the chips in and out, both to replace them when one chip needsto be discarded and to replace them by another chip in order to performa different type of test, the microfluidic chips can be incorporated ina pluggable module referred hereafter as “cartridge”. A cartridgecomprises at least a microfluidic chip and a mechanical apparatus toinsert the chip into the diagnostic device. The mechanical apparatuscould for example be a rail combined with a locking system. The lockingsystem does not need to be user-operated, it can simply click into placeso that a certain amount of force is necessitated to remove thecartridge and so that the cartridge is stabilized in its position.Because signals are detected with an imager, the locking does not needto be extremely precise, as small displacements can be easily correctedby the software or by the chip hardware by performing basic patternrecognition.

The cartridge can contain further elements, such as the exemplaryelements illustrated below:

An electronic chip that connects to the analytical device once thecartridge is inserted. This electronic chip could store informationabout the microfluidic chip carried by the device, such as type ofmicrofluidic chip, as well as the sequence of tasks, such as actuations,detections, heat cycles etc. that need to be performed by the analyticaldevice in order to perform a given test (e.g. a diagnostic test). If achip is contained on the cartridge it should also store preemption dateof the cartridge.

A temperature control system. As mentioned above the temperature controlcan be partially incorporated in the microfluidic chip itself, forexample in the form of resistors imbedded in the PDMS matrix. However,the temperature control in the form of heaters and/or Pelletierjunctions can also be incorporated in the cartridge but outside themicrofluidic chip itself. In some embodiments, this solution might bemore costly than incorporating it in the analytical device (but outsidethe cartridge), because it will be discarded every time the cartridgewill be discarded. However this configuration will be beneficial ifdifferent types of microfluidic chips need very different temperaturecontrol schemes.

A set of optics, in particular filters. In order to make the diagnosticdevice compatible with different sets of dies that emit light atdifferent wavelengths, the filter (4) (see FIG. 1) should be adapted.One way to do this is to incorporate the filter into the cartridge sothat it gets swapped together with the microfluidic circuit. It is alsouseful to have the filter on the cartridge if it is not filling theentire area, but leaving some areas free so that part of themicrofluidic chip can be imaged for monitoring of fluid progression.That configuration might change from microfluidic chip to microfluidicchip.

Incorporating a temperature control system and/or optics into thecartridge, makes the cartridge much more expensive. In order to make thediagnostic device modular, that is filters and heating systems that canbe swapped to accommodate different microfluidic chips, a secondswappable module (hereinafter referred to as the module) can beintroduced that incorporates an optical filter and/or a temperaturecontrol system and possibly other functionalities. The cartridge will bediscarded every time a microfluidic chip needs to be replaced, but themodule will only be swapped when the type of tests performed or the typeof cartridges being used necessitate the change. Moreover the modulewill be reusable, so that of the previous series of tests needs to beperformed again it can be swapped back in.

When a different type of cartridge or a different module is being used,the sequence of tasks that need to be performed by the diagnostic toolwill change. For example the position of the reactors on themicrofluidic chip might change, so that the relevant data has to becollected from different parts of the imager. The summation circuit ofFIG. 7, the microcontroller and other electronics then should bereprogrammed. Also the actuation scheme and the temperature controlschemes might change and need to be reconfigured etc. Thisreconfiguration of the device can be achieved by various means includingthe following exemplary means:

The program can be stored on a chip located on the cartridge or on themodule and then downloaded by the other elements of the device.

A memory element in the analytical device (on the imager chip or on aseparate chip) can store the programs for all the compatible cartridges.The cartridge then only needs to be identified in order to activate theprogram. Identification of the cartridge could be obtained by means of asimple chip on the cartridge (that now only needs to store the type ofcartridge instead of an entire program) or by RF-ID contained in thecartridge. The user could dial in the type of cartridge, either bytyping it on a computer with which the device communicates, or directlyin the device if such an I/O functionality is integrated (for example bysetting a series of switches to dial in a cartridge code).

The program could be downloaded from a computer, or directly from theinternet, depending on how the analytical device is configured.

Hybrid solutions. For example, all the programs for the compatiblecartridges at the time of manufacturing could be stored in theanalytical device. If a new type of cartridge is released, or if thedevice is upgraded to be made compatible with other cartridges (thiscompatibility does not need to be due to physical reasons, it can belinked to marketing strategy), the corresponding program could be addedto the databank by downloading it from a computer, from the internet, orfrom a pluggable device (such as a USB memory device). The chipcontaining the programs could also be swapped out, either at a servicecenter or at the end users home if an adequate mechanism is built in.

If the device is marketed with a market segmentation strategy all therelevant programs can be stored in the chip, but the use of certain typeof cartridges artificially deactivated. Upgrading the device, forexample by paying a fee on the Internet portal, could then activate thisfunctionality.

Further mechanisms can be incorporated into this analytical device byincorporating a stripped down GPS system and only allowing it to operatein certain region of the planet, or by monitoring the location throughthe Internet by checking the IP number. It could also be required forthe end-user to log in on the portal in order to make the deviceoperable or in order to access the data. Those mechanisms might beparticularly suitable in applications wherein the analytical device isused for medical purposes (e.g. diagnostic purposes).

In some embodiments, the frame of the diagnostic device can be designedin such a way that when the cartridge is inserted into the device, theflap is automatically pushed down, as shown in FIG. 15. Thisconfiguration has the further advantage of simplifying the sealing ofthe flab to the bulk part of the microfluidic chamber. In theconfiguration fo FIG. 15, the element (66) that pushes down the flap canalso contain a heater or other additional sealing mechanisms intended toseal the microfluidic chip. However, PDMS tends to naturally stick toPDMS so that the pressure exerted on the flap by (66) can be sufficientto seal adequately the chamber without further actuation. The pressureis simply created by the tight fit of the cartridge within the frame ofthe diagnostic device. Optionally, element (66) can be actuated to pushdown once the cartridge is completely introduced. Element (66) is shownwith rounded edges so as to avoid damaging the microfluidic chip whenintroduced into the diagnostic device.

In FIG. 15, element (60) is the frame of the diagnostic device, element(61) is the rail system on the diagnostic device, element (62) theclip-in mechanism on the diagnostic device, element (63) the clip-inmechanism on the cartridge, element (64) the rail system on themicrofluidic device, element (65) the substrate of the cartridge andelement (66) the element that pushes down the flap. The rail system andclipping system can be equipped with a mechanism to detect the“clipped-in” condition, such as for example an electrical connectionfrom one rail system to the other that is close when clipped-in, or aswitch on the analytical device that is actuated when the cartridge isintroduced. When the cartridge is introduced, the programmed sequence oftasks can start, for example, the flap can be thermally sealed or thepiston (54) can push onto the chamber (55) to pressurize the biologicalsample and push it into the microfluidic channel.

The analytical device can comprise a sterilization mechanism that willbe activated upon removal of the microfluidic cartridge. Thissterilization mechanism also destroys the residual bio-molecules in thedevice. It can be implemented for example by heating up the interior ofthe apparatus to high temperature.

It is to be understood that the disclosure are not limited to particularconfigurations or the device, samples, applications or systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a,” “an,” and “the” includeplural referents unless the content clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the disclosure pertains. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the device(s) and methods herein disclosed,specific examples of appropriate materials and methods are describedherein.

The description set forth above is provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the devices, systems and methods of thedisclosure, and are not intended to limit the scope of the disclosure.Modifications of the above-described modes for carrying out thedevice(s) and methods herein disclosed that are obvious to persons ofskill in the art are intended to be within the scope of the followingclaims. All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

A number of embodiments of the device(s) and methods herein disclosedhave been described. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe disclosure. Accordingly, other embodiments are within the scope ofthe following claims.

1.-13. (canceled)
 14. A method for pressurizing a microfluidic chipcomprising sealed fluidic circuits, comprising: providing a firstchamber in the microfluidic chip; and applying pressure to the firstchamber.
 15. The method of claim 14, further comprising providing asecond chamber in the microfluidic chip, so that pressure applied to thefirst chamber is transferred to the second chamber.
 16. The method ofclaim 15, wherein the second chamber is a reactant chamber.
 17. Themethod of claim 15, wherein pressure is applied through a mechanicalactuator and a piston.
 18. The method of claim 17, wherein sacrificialmembranes or sealed valves are connected to the microfluidic chip, themicrofluidic chip being located between the piston and the sacrificialmembranes or sealed valves.
 19. The method of claim 18, wherein themechanical actuator is programmed to increment pressure exerted on thefirst chamber by predetermined amounts, and the mechanical actuator canbe continuously tunable.
 20. The method of claim 14, further comprising:providing a second chamber; connecting the second chamber with themicrofluidic chip, the first chamber connected to a first set ofchannels of the microfluidic chip, the second chamber connected to asecond set of channels of the microfluidic chip; and applying pressureto second chamber, wherein pressure to the first and second chamber isapplied through different actuators.
 21. The method of claim 14, whereinthe microfluidic circuit is programmable through insertion in acartridge. 22.-25. (canceled)